Indoline-5-Sulfonamides: A Role of the Core in Inhibition of Cancer-Related Carbonic Anhydrases, Antiproliferative Activity and Circumventing of Multidrug Resistance

The overexpression and activity of carbonic anhydrase (CA, EC 4.2.1.1) isoforms CA IX and CA XII promote the accumulation of exceeding protons and acidosis in the extracellular tumor environment. Sulfonamides are effective inhibitors of most families of CAs. In this study, using scaffold-hopping, indoline-5-sulfonamide analogs 4a–u of the CA IX-selective inhibitor 3 were designed and synthesized to evaluate their biological properties. 1-Acylated indoline-5-sulfonamides demonstrated inhibitory activity against tumor-associated CA IX and XII with KI values up to 132.8 nM and 41.3 nM. Compound 4f, as one of the most potent inhibitors of CA IX and XII, exhibits hypoxic selectivity, suppressing the growth of MCF7 cells at 12.9 µM, and causes partial inhibition of hypoxia-induced CA IX expression in A431 skin cancer cells. 4e and 4f reverse chemoresistance to doxorubicin of K562/4 with overexpression of P-gp.


Introduction
One of the factors of the reduced effectiveness of chemo-and radiotherapy is hypoxic regions of tumors [1][2][3]. The deficient oxygen supply leads to the activation of transcriptional factor HIF-1α in tumor cells. In its turn, HIF-1α conceives expression of many proteins that advantage the development of aggressive phenotype of cancer cells [4]. One of the features of hypoxic cancer cells is the acidic extracellular pH (pHe) [5]. Increased level of protons causes resistance to the widely applied chemotherapeutic agents, including doxorubicin (Dox) [6] and cisplatin [7]. Aside from resistance development, acidic pHe destructs extracellular matrix and declines cell adhesion resulting in invasion and metastasis of tumors [8,9].
The acidic extracellular tumor environment arises from the coordinated work of transmembrane carbonic anhydrase (CA) IX and CA XII with sodium-hydrogen exchanger-1 [10]. CA IX and CA XII genes have a hypoxia response element that indicates their activation under the control of HIF-1α [11]. CA IX and CA XII are among 13 zinc metalloproteins of the α-carbonic anhydrases family with enzymatic activity [12]. Both enzymes are anchored in the cell membrane with active sites located in the extracellular milieu [13]. Utilizing the Zn 2+ ion and His residues, CA IX and CA XII catalyze the hydration of CO 2 to HCO 3 − and H + [14]. CA IX presence in normal tissue is limited to GI mucosa, whereas its expression enzymes are anchored in the cell membrane with active sites located in the extracellular milieu [13]. Utilizing the Zn 2+ ion and His residues, CA IX and CA XII catalyze the hydration of CO2 to HCO3 − and H + [14]. CA IX presence in normal tissue is limited to GI mucosa, whereas its expression in tumor cells is widespread and found in highly aggressive types of cancer, including breast, lung, brain, colon, rectum and kidney tumors [12,15]. Numerous studies have shown that the expression of CA IX is the prediction of a poor outcome [16][17][18][19]. However, CA XII, along with expression in several types of tumors, was also found in breast and colon normal tissues [20,21] and can correlate with a positive [22] as well as a negative prognosis [23,24]. Despite such uncertainty, several studies demonstrated the significance of CA XII in cancer severity [25,26]. For example, CA XII expression was increased in chemoresistant cells and influenced P-glycoprotein (P-gp) activity [27]. The development of CA XII as a pharmacological target for cancer therapy has led to successful reports of selective CA XII inhibitors overcoming multidrug resistance (MDR) in vivo [28,29].
Coordinating with the Zn 2+ ion, sulfonamides such as acetazolamide (1) and SLC-0111 (2) (Figure 1) are the most studied inhibitors of the CA enzyme family [30][31][32][33][34][35][36][37][38]. SLC-0111 is the selective CA IX inhibitor; it reduces acidic pH, leading to the tumor and metastasis regression in preclinical models [39][40][41]. Combinations of different chemotherapeutic agents with SLC-0111 demonstrate a high potential for CA IX inhibition to treat aggressive and resistant types of cancer [42][43][44]. For example, the combination of the SLC-0111 with gemcitabine increased survival and enhanced tumor cell death of highly hypoxic and resistant pancreatic ductal adenocarcinomas in vivo [45]. Different groups have reported successful approaches to inhibit cancer-related CA IX and CA XII by indole-based sulfonamides [46][47][48]. Earlier, we studied 1-substituted isatin-5-sulfonamides as new CAs inhibitors, which suppressed the growth of tumor cells at low micromolar concentrations [49]. At this stage, we continue the work on modifying the structure of 5-indanesulfonamide derivative 3, which selectively inhibits CA IX [50] and enhances the therapeutic effect of tumor irradiation in vivo [51]. However, 1-aminoindane-5-sulfonamide 3 has a limited antiproliferative effect on tumor cells [49], so the development of carbonic anhydrase inhibitors with improved anticancer potencies is a pivotal task. Scaffold hopping is an effective method to discover novel biologically active compounds by central core modification of the known lead compound [52][53][54][55][56]. Based on the selectivity of 3 toward CA IX and the known antiproliferative effect of indoline derivatives [57][58][59], we applied the scaffold hopping approach to replace 1-aminoindane to indoline core 4, varying the structure of hydrophobic tail for the best binding to the targets ( Figure 2).
To examine the results of the scaffold hopping for the discovery of new carbonic anhydrase inhibitors, we docked indane-5-sulfonamide 3 with indoline-5-sulfonamide analog 4 in the active site of CA IX (Figure 3). According to the modeling, 3 and perfluoro benzoyl derivative 4 demonstrate a similar orientation in the active site of CA IX. Both ligands interact with Zn 2+ ions as sulfonamidate anions and form identical hydrogen bonds with Thr 199 and arene-H interactions with Leu 198 residues. The distinction of Different groups have reported successful approaches to inhibit cancer-related CA IX and CA XII by indole-based sulfonamides [46][47][48]. Earlier, we studied 1-substituted isatin-5-sulfonamides as new CAs inhibitors, which suppressed the growth of tumor cells at low micromolar concentrations [49]. At this stage, we continue the work on modifying the structure of 5-indanesulfonamide derivative 3, which selectively inhibits CA IX [50] and enhances the therapeutic effect of tumor irradiation in vivo [51]. However, 1-aminoindane-5-sulfonamide 3 has a limited antiproliferative effect on tumor cells [49], so the development of carbonic anhydrase inhibitors with improved anticancer potencies is a pivotal task. Scaffold hopping is an effective method to discover novel biologically active compounds by central core modification of the known lead compound [52][53][54][55][56]. Based on the selectivity of 3 toward CA IX and the known antiproliferative effect of indoline derivatives [57][58][59], we applied the scaffold hopping approach to replace 1-aminoindane to indoline core 4, varying the structure of hydrophobic tail for the best binding to the targets ( Figure 2).   To examine the results of the scaffold hopping for the discovery of new carbonic anhydrase inhibitors, we docked indane-5-sulfonamide 3 with indoline-5-sulfonamide analog 4 in the active site of CA IX (Figure 3). According to the modeling, 3 and perfluoro benzoyl derivative 4 demonstrate a similar orientation in the active site of CA IX. Both ligands interact with Zn 2+ ions as sulfonamidate anions and form identical hydrogen bonds with Thr 199 and arene-H interactions with Leu 198 residues. The distinction of indoline ligand 4 from indane 3 lies in the orientation of the perfluorobenzoyl group that allows indoline inhibitor 4 to form novel interactions with the amino acid residue of CA IX. Thus, the additional hydrogen bond between the amides group of indoline ligand 4 and Gln 92 and the similar binding mode of central cores point to a promising potential of 1-acylindoline-5-sulfonamides 4 as new CA IX inhibitors.
indoline ligand 4 from indane 3 lies in the orientation of the perfluorobenzoyl group that allows indoline inhibitor 4 to form novel interactions with the amino acid residue of CA IX. Thus, the additional hydrogen bond between the amides group of indoline ligand 4 and Gln 92 and the similar binding mode of central cores point to a promising potential of 1-acylindoline-5-sulfonamides 4 as new CA IX inhibitors.

Chemistry
Indolines exhibit chemical properties close to N-substituted anilines. Developed methods of indole reduction and reverse dehydrogenation open the convenient synthetic way to 5-substituted indoles. In particular, Terent'ev and Preobrazhenskaya applied this method to obtain indole-5-sulfonamide [60]. Firstly, to carry out an electrophilic substitution, indoline (5) was protected with acetic anhydride in quantitative yield (Scheme 1). Next, 1-acetylindoline (6) was treated with chlorosulfuric acid, which led to 1-acetylindloine-5-sulfochloride (7) in a good yield (81%). The following interaction of sulfochloride 7 with ammonia in THF resulted in the formation of 1-acetylindoline-5-sulfonamide (8) with an 89% yield. In the next step, hydrolysis of indoline 8 by hydrochloric acid gave indoline-5-sulfonamide (9) with 81% yield. To synthesize a library of 1-acylindoline-5-sulfonamides 4, indoline-5-sulfonamide 9 was acylated by a series of acyl chlorides in the presence of pyridine in CHCl3 (Scheme 2). For the synthesis of isonicotinic derivative 4q, the corresponding acid was activated by CDI and then treated with indoline-5-sulfonamide 9 in the presence of DMAP in THF.  To synthesize a library of 1-acylindoline-5-sulfonamides 4, indoline-5-sulfonamide 9 was acylated by a series of acyl chlorides in the presence of pyridine in CHCl 3 (Scheme 2). For the synthesis of isonicotinic derivative 4q, the corresponding acid was activated by CDI and then treated with indoline-5-sulfonamide 9 in the presence of DMAP in THF. Applying these procedures, a library of 21 1-acylindoline-5-sulfonamides 4a-4u was synthesized with 50-79% yields. Scheme 1. The synthesis of indoline-5-sulfonamide 9.

Carbonic Anhydrase Inhibition Assay
Synthesized indoline-5-sulfonamide derivatives 4a-4u, 10 were evaluated against hCA I, hCA II, hCA IX and hCA XII by a stopped-flow technique (Table 1). * Mean from three different assays, by a stopped-flow technique (errors were in the range of ± 5-10% of the reported values); ** K I data for compound 3 taken from previous research [50]; N.T.-not tested.
CA I and CA II are cytosolic proteins, that are widely expressed in erythrocytes, the eye, the GI tract, osteoclasts, and kidney cells [61]. CA II inhibitors are used in the clinical setting as diuretics and for glaucoma-related intraocular hypertension [62]. At the same time, CA I and CA II are considered the main off-target isoforms for the development of cancer-related CA IX and CA XII inhibitors [63].
The majority of indoline-5-sulfonamides 4a-4u, 10 inhibits CA I at concentrations less than 100 nM. Structure-activity relationship points out that derivatives of benzoic acids with chlorine atoms 4f, g, h demonstrate lower affinity toward this off-target isoform. At the same time, replacement of the phenyl 4a by pyridine 4q, thiophene 4r or cycloalkanes 4s, t, u does not affect K I notably.
3-Chlorophenyl 4f, thiophene 4r and cyclopentyl 4s, t derivatives are the most potent derivatives inhibiting CA IX around 100 nM. These compounds outperform a group of the least active derivatives by more than 100 times, which indicates the high importance of the structure of the lipophilic fragment for CA IX inhibition. Additionally, a comparison of indane 3 with indoline analog 4e reveals that 1-aminoindane scaffold appears as a preferred scaffold toward CA IX isoform. Thereby, the affinity of 1-acylindoline-5-sulfonamides 4a-u for CA IX mainly depends on the structure of acyl moiety and is notably enhanced by 3-chlorophenyl and five-membered rings of aromatic and saturated nature.
1-Acylated indoline-5-sulfonamides 4d, g, h are potent inhibitors of CA XII. Notably, for indoline-5-sulfonamides, the introduction of acyl group (4a), compared to less polar alkyl fragment (10), leads to a clear gain of activity against CA XII. The majority of 1-Pharmaceuticals 2022, 15, 1453 6 of 20 acylated indoline-5-sulfonamides inhibit CA XII around 100 nM. Additionally, 4-chloro 4g and 3,4-dichloro derivatives 4h demonstrate selectivity over CA I and CA IX isoforms and have similar K I with CA II. Based on the CA inhibition profile, 3-chlorobenzoyl derivative 4f, as one of the most potent compounds toward CA IX and CA XII, was selected as the lead compound for further investigation.

Docking Studies
To investigate the difference of K I against CA IX in 1-acylindoline-5-sulfonamides, we docked compound 4f (Figure 4) in the active site of CA IX and compared it to a model of 4e. As in the case with perfluoro derivative 4e, 4f displays coordination with Zn 2+ ion as a sulfonamidate anion. Additionally, both ligands are acceptors of hydrogen bonds of Thr 199 residue. The differences between 4e and 4f occurred in interactions of indoline and acyl fragments. In particular, ligand 4f, compared with 4e, lacks arene-H interaction with Leu 198 but interacts with Gln 67 instead of Gln 92. Almost 10-fold higher activity of 4f in vitro may be explained by the strength of interactions and physicochemical properties of compounds.
of indane 3 with indoline analog 4e reveals that 1-aminoindane scaffold appears as a preferred scaffold toward CA IX isoform. Thereby, the affinity of 1-acylindoline-5-sulfonamides 4a-u for CA IX mainly depends on the structure of acyl moiety and is notably enhanced by 3-chlorophenyl and five-membered rings of aromatic and saturated nature.
1-Acylated indoline-5-sulfonamides 4d, g, h are potent inhibitors of CA XII. Notably, for indoline-5-sulfonamides, the introduction of acyl group (4a), compared to less polar alkyl fragment (10), leads to a clear gain of activity against CA XII. The majority of 1acylated indoline-5-sulfonamides inhibit CA XII around 100 nM. Additionally, 4-chloro 4g and 3,4-dichloro derivatives 4h demonstrate selectivity over CA I and CA IX isoforms and have similar KI with CA II. Based on the CA inhibition profile, 3-chlorobenzoyl derivative 4f, as one of the most potent compounds toward CA IX and CA XII, was selected as the lead compound for further investigation.

Docking Studies
To investigate the difference of KI against CA IX in 1-acylindoline-5-sulfonamides, we docked compound 4f (Figure 4) in the active site of CA IX and compared it to a model of 4e. As in the case with perfluoro derivative 4e, 4f displays coordination with Zn 2+ ion as a sulfonamidate anion. Additionally, both ligands are acceptors of hydrogen bonds of Thr 199 residue. The differences between 4e and 4f occurred in interactions of indoline and acyl fragments. In particular, ligand 4f, compared with 4e, lacks arene-H interaction with Leu 198 but interacts with Gln 67 instead of Gln 92. Almost 10-fold higher activity of 4f in vitro may be explained by the strength of interactions and physicochemical properties of compounds.

Antiproliferative Activity of Indoline-5-sulfonamides
A series of synthesized indoline-5-sulfonamides was tested on the MCF7 cancer cell line under normoxia and hypoxia using the MTT test. According to the results presented in Table 2, indoline-5-sulfonamides demonstrate a moderate antiproliferative effect. In

Antiproliferative Activity of Indoline-5-sulfonamides
A series of synthesized indoline-5-sulfonamides was tested on the MCF7 cancer cell line under normoxia and hypoxia using the MTT test. According to the results presented in Table 2, indoline-5-sulfonamides demonstrate a moderate antiproliferative effect. In general, indoline-5-sulfonamides do not lose their activity under hypoxia, which is the common reason for resistance to current antitumor agents. Comparing MTT results of 3 and indoline analog 4e, we observed a clear gain of antiproliferative activity for the indoline derivative against the MCF7 line in normoxia and hypoxia. Again, indoline-5-sulfonamide 4f as the most potent compound against MCF7 cells under hypoxia conditions (IC 50 = 12.9 µM) demonstrates two-fold higher activity compared to normoxia conditions.
To evaluate the influence of CA inhibition on the antiproliferative activity, we selected five compounds 4b, f, m, n and 10 with different inhibition profiles against CA IX for in-depth testing on cancer cells with increased CA IX expression. The database www. proteinatlas.org (accessed on 13 October 2022) contains data on gene expression in various cell lines. According to the data obtained from this database, A431 cells express CA IX at a very high level, which distinguishes them from human cell lines belonging to other tissues [https://www.proteinatlas.org/ENSG00000107159-CA9/cell+line (accessed on 13 October 2022)]. A431 cells are skin cancer cells, and their growth has already been intensely analyzed in hypoxia [65][66][67]. Hypoxia significantly alters the activity of signaling pathways in these cells; some of the revealed changes may be related to the induced activity of CAs. Moreover, Ren and colleagues described that hypoxia modulates cellular pathways in A431 cells, which are associated with radioresistance and enhanced migration [65]. Given the observations described above, we were expecting to reveal the high antiproliferative activity of the selected compounds against skin cancer cells. Surprisingly, the activity turned out to be at a moderate level. The cell growth curves are shown in Figure 5A-E. As can be seen in normoxia, the compounds inhibit cell growth by no more than 20%. Compounds 4b and 4m were the least active in normoxia; this finding may be associated with their low activity against CA IX (Table 1). In hypoxia, the activity of tested compounds was increased, but the 50% inhibition of growth was never achieved. than 20%. Compounds 4b and 4m were the least active in normoxia; this finding may be associated with their low activity against CA IX (Table 1). In hypoxia, the activity of tested compounds was increased, but the 50% inhibition of growth was never achieved. Compound 4f was the most active under hypoxic conditions, inhibiting the growth of A431 cells by 44%. We wondered whether compound 4f affected CA IX expression or whether its activity was limited to inhibiting the enzyme activity reported above (Table  Compound 4f was the most active under hypoxic conditions, inhibiting the growth of A431 cells by 44%. We wondered whether compound 4f affected CA IX expression or whether its activity was limited to inhibiting the enzyme activity reported above (Table 1). To induce CA IX expression, cells were placed in hypoxia for 24 h, and then protein expression was analyzed by immunoblotting. Because the data on the expression of hypoxia-related proteins in the literature are highly inconsistent, we introduced additional controls. Antibodies to hypoxia-regulated and "non-hypoxic" proteins were used [68,69]. The expression of kinase Akt, which is not regulated directly by hypoxia/HIF-1α, was not altered under hypoxia conditions ( Figure 5F). On the contrary, the expression of proteins (CA IX, PD-L1, GLUT1) associated with hypoxia pathways was significantly increased. Thus, these data again support a high expression of CA IX in A431 cells. In A431 cells treated with compound 4f, a moderate decrease in CA IX expression was observed, whereas the expression of hypoxia-regulated proteins PD-L1 and GLUT1 did not change. Thus, lead compound 4f not only blocks the enzyme activity of CA IX, but also causes partial inhibition of hypoxia-induced CA IX expression in skin cancer cells.
Next, we examined the activity of the whole series of indoline-5-sulfonamides against leukemia cell line K562 . Surprisingly, among all tested compounds, only perfluoro derivative 4e demonstrated significant activity and inhibited the growth of the cells at 10 µM.
Other indoline derivatives showed low activity against K562 cells at 50 µM or the highest concentrations.
Earlier, Kopecka and colleagues have shown that CA XII can interact with P-gp and influence its activity [27]. Subsequently, silencing or inhibition of CA XII by chemical agents may restore the sensitivity to Dox of resistant K562 and other tumor cells [70,71]. Given the potent inhibition of 4f against CA XII and the activity of 4e against K562, we examined their ability to reverse the chemoresistance of K562/4 with P-gp to Dox. MTT-test results summarized in Figure 6 show that

Discussion
Scaffold hopping makes it possible to reveal previously unknown data on the structure-activity relationships. It is especially important for the search for selective inhibitors

Discussion
Scaffold hopping makes it possible to reveal previously unknown data on the structureactivity relationships. It is especially important for the search for selective inhibitors of enzymes with off-target isoforms. In addition to a Zn 2+ binding sulfonamide group, inhibitors of tumor-associated CA IX require a lipophilic fragment responsible for interaction with the hydrophobic side of the enzyme. Thus, in the structure of the CA IX-selective sulfonamide 3, we replaced the 1-aminoindane with an indoline fragment and varied the hydrophobic tail to create new inhibitors of CA IX. As a result, we have obtained a broad series of 1-substituted indoline-5-sulfonamides exhibiting a broad spectrum of K I against four CA isoforms. In general, replacement of 1-aminoindane scaffold with indoline led to a significant increase of activity against cytosolic CA I and CA II isoforms. Simultaneously, inhibition of CA IX and CA XII by 1-acylindoline-5-sulfonamides 4a-4u can vary in the range of two orders. Among indoline-5-sulfonamides with a high affinity toward cancerrelated isoforms, indoline 4f demonstrated a decreased activity toward cytosolic CA I and CA II.
The antiproliferative activity of the indoline-5-sulfonamides 4a-4u series indicated the good activity of potent CA IX and CA XII inhibitor 4f, which exhibits hypoxic selectivity and inhibits the growth of MCF7 cells at 12.9 µM. At the same time, indoline analog 4e outperforms indane analog 3 against the MCF7 line. MTT-test of chosen indoline-5sulfonamides did not reveal a high antiproliferative activity against A431 cells with a high expression of CA IX. However, immunoblotting has shown that the lead 4f not only inhibits CA IX, but also suppresses CA IX expression under hypoxic conditions in A431 skin cancer cells.
The good inhibitory activity of some indoline-5-sulfonamides against CA XII, a new target for overcoming MDR, made it compelling to study 4e and 4f ability to overcome the resistance of K562/4 cells with the expression of P-gp. Combinations of compounds 4e and 4f with Dox pointed to their ability to increase the suppression of resistant cells K562/4.
Overall, 1-acylated indoline-5-sulfonamides represent a new scaffold of nanomolar inhibitors and suppressors of tumor-associated CA IX and CA XII that points out their potential as adjuvant and MDR-overcoming agents (Supplementary Materials).

Instruments and General Information
NMR spectra were recorded on a Varian VXR-400 instrument operated at 400 MHz ( 1 H NMR) and 100 MHz ( 13 C NMR). Chemical shifts were measured in DMSO-d6, using tetramethylsilane as an internal standard. Analytical TLC was performed on Silica Gel F254 plates (Merck), column chromatography with a SilicaGel Merck 60. Melting points were determined using a Buchi SMP-20 apparatus and were uncorrected. High-resolution mass spectra were recorded with electron spray ionization on a Bruker Daltonics microOTOF-QII instrument. HPLC was performed using a Shimadzu Class-VP V6.12SP1 system. IR spectra were recorded on a Nicolet iS10 Fourier transform IR spectrometer (Nicolet iS10 FT-IR, Madison, WI). All solvents, chemicals and reagents were obtained commercially and used without purification. The purity of final compounds 4a-4u, 10 was ≥95% as determined by HPLC analysis.

1-Acetyl-5-(chlorosulfonyl)indoline (7)
To a stirring chlorosulfonic acid (15 mL, 0.225 mol) cooled in an ice bath was added N-acetylindoline (6, 5.4 g, 0.034 mol) portionwise at 0-5 • C. The resulting mixture was heated to 50 • C for 2 h. Upon completion, the reaction mixture was cooled and poured onto ice, and the precipitate was filtered and washed with cold water twice to obtain 7.0 g (81%)  To a solution of isonicotinic acid (90 mg, 0.73 mmol) in THF (5 mL) was added CDI (119 mg, 0.73 mmol). After 30 min until the end of the release of bubbles, a solution of indoline-5-sulfonamide (9, 110 mg, 0.6 mmol) in THF (5 mL) was added dropwise. The resulting mixture was heated to 40 • C for 2 h, cooled, and the solvent was removed in vacuo. The residue was diluted with water (7 mL) and filtrated. The crude product was crystallized from EtOH to get 84 mg (50%) of pure 1-isonicotinoylindoline- 5

CA Inhibitory Assay
The CO 2 hydration activity of the four hCA isoforms was monitored using an Applied Photophysics stopped-flow instrument [73]. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM HEPES (pH 7.4) as a buffer, and 20 mM NaClO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10-100 s. To determine the kinetic parameters by Lineweaver-Burk plots and the inhibition constants, a concentration of CO 2 between 1.7 to 17 mM was used. At least six measurements of the original 5-10% reaction were used to assess the initial velocity for each inhibitor. The uncatalyzed rates were determined and detracted from the total observed rates. Stock inhibitor solutions (10-100 mM) were prepared in distilled-deionized water, and dilutions up to 0.1 nM were done with the buffer test. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay in order to allow for the formation of the E-I complex or for the eventual active site-mediated hydrolysis of the inhibitor. The inhibition constants were obtained by non-linear least-squares methods using PRISM 6 and the Cheng-Prusoff equation, as reported earlier [74][75][76], and represent the mean from at least three different determinations. All enzymes were recombinant proteins obtained in-house, and their concentration in the assay system was 4.5-12 nM.

Molecular Modelling Studies
Molecular modelling was performed using Molecular Operating Environment (MOE) version 2014.09; Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2014. CA IX structure was read from a PDB file 5sz5. Structural issues were automatically corrected using the Structure Preparation application. The hydrogen bond network and charges were optimized. Tethered energy minimization was performed using an AMBER10:EHT force field. The binding pocket of the receptor was specified by proximity to the cocrystallized ligand atoms. Chosen compounds were prepared using the wash command, and then partial charges were calculated. Ligand's energy minimization was done using an MMFF94x force field. Deprotonation of strong acids and protonation of strong bases were checked in the wash panel. Docking placement was done using the triangle matcher algorithm with the 'rotate bonds' option. The 1st scoring function was London dG, and the 2nd scoring function was GBVI/WSA dG. MOE-Dock performed 30 independent docking runs. Docked complexes were ranked based on the docking scores (S). Finally, predicted complexes were analyzed for molecular interactions using the MOE window.

Cells and Antiproliferative Evaluation
The MCF7 human breast, A431 human skin cancer cells and K562 (ATCC CCL-243) chronic myelogenous leukemia cells were obtained from the ATCC collection. The MDR subline K562/4 [77] (kind gift of Dr. Alexander Shtil, Blokhin N.N. National Medical Research Center of Oncology) was obtained by stepwise selection of K562 cells for survival under continuous exposure to Dox. This subline expresses the MDR1 gene and functional P-gp and is characterized by a high resistance index for Dox [78]. The MCF7 and A431 cells were cultured in standard 4.5 g/L glucose DMEM medium (Gibco) supplemented with 10% FCS (HyClone), 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin (PanEco), 100 µg/mL sodium pyruvate (Santa Cruz) at 37 • C, 5% CO 2 and 80-85% humidity in Nu-Aire incubator. Suspension myelogenous leukemia cells (K562, K562/4) were propagated in RPMI-1640 (PanEco) with 5% FCS (HyClone), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C, 5% CO 2 , and 80-85% humidity in Binder incubator. Cells in the logarithmic phase of growth were used in the experiments. The growth inhibitory activity of compounds was assessed by the MTT test based on the metabolism of the MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Applichem) in living cells, with modifications as described previously in [49]. Compounds at different concentrations in 100 µL of the appropriate medium were added, and the cells were grown for 72 h. The hypoxia (1% O 2 ) conditions were simulated in Binder multigas incubator, as described in [49]. After incubation with compounds, the medium was removed, the MTT reagent that was dissolved in the medium was added to the final concentration of 0.2 mg/mL to each well, and the incubation was performed for 2 h. Then the cell supernatants were removed, and purple formazan crystals were dissolved in 100% DMSO (350 µL per well). Culture plates were gently shaken, and the absorbance was measured at 571 nm with a reference wavelength of 630 nm on a MultiScan reader (ThermoFisher, Waltham, MA, USA). The viability of the cells was expressed as a percentage of the control. Dose-response curves were analyzed by regression analysis using sigmoid curves (Log(concentration) vs. normalized absorbance).

Statistical Analysis
All results were reported as means ± SD. One-way analysis of variance was used for the analysis of data. Differences were defined as significant at p < 0.05. GraphPad Prism7 (GraphPad Software, San Diego, CA, USA) was used for the determination of the half-maximal inhibitory concentration (IC 50 ) values.

Immunoblotting
A431 cells were seeded on 100 mm dishes (Corning, Corning, NY, USA), and after 24 h growth, the compound 4f was added to a fresh medium. To prepare cell extracts, A431 cells were twice washed in phosphate buffer and incubated for 10 min on ice in the modified lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.5% Igepal CA-630, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM sodium orthovanadate and aprotinin, leupeptin and pepstatin (1 µg/mL each) as described earlier in the work [79]. The protein content was determined by the Bradford method.
A431 cell lysates were separated in 10% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane (GE HealthCare, Chicago, IL, USA), and processed according to a standard protocol. Akt, GLUT1, PD-L1, and CA IX antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); the antibodies against GAPDH (Cell Signaling Technology) were added to standardize loading. Goat anti-rabbit IgGs (Jackson ImmunoResearch, West Grove, PA, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Signals were detected using the ECL reagent as described in Mruk and Cheng's protocol [80] and an ImageQuant LAS4000 system (GE HealthCare).